The central theme of our Botanical Center is to enhance safety and health benefits of botanical dietary supplements that are widely used by U.S. women. In the previous funding periods, Project 1 has pioneered the development of phytoanalytical methods for botanicals and supplied rigorously standardized extracts for three Phase I or II clinical trials. Continuing the close interdisciplinary collaboration with all Center Projects and Cores, Project 1 will show that the complex chemical composition of plant metabolomes can be (1) selectively controlled to generate unique modulated extracts, (2) broadly monitored to achieve multi-target standardization, and (3) systematically explored to better comprehend the topology of botanical markers. Focusing on the complex herbal metabolome, Project 1 hypothesizes that metabolomic analysis enables multi-target standardization and holistic assessment of botanicals. Considering our recent progress with three well-studied plants, we hypothesize that innovative phytochemistry can resolve and preparatively control metabolomic mixtures, make metabolomic standardization feasible, and unravel previously unknown/low-level metabolites. In three Specific Aims, we will fully develop the countercurrent-based method of chemical subtraction to generate libraries and quantities of knock-out, knock-in, and other phytochemically controlled extracts (DESIGNER extracts) for biological studies in the Center (Aim 1). To make metabolomic, multi-target standardization more practical, we will develop primary analytical methods that utilize NMR to eliminate the widespread dependence of LC methods on identical reference standards (Aim 2). Systematic mining of metabolomic chemodiversity (metabolomic mining) will uncover new primary and secondary metabolites for development as potential markers in the Center context. This effort in Aim 3 will include compounds of natural deep eutectic solvents (NADES) which are abundant but underexplored constituents of botanical extracts. The long-term objective of Project 1 is to advance the understanding of botanical quality control measures and broaden the base for chemical and biological standardization of botanicals.